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Plant Cell, Tissue and Organ Culture (PCTOC) https://doi.org/10.1007/s11240-018-1453-2
CORRECTION
Correction to: Shoot regeneration process and optimization of Agrobacterium-mediated transformation in Sinningia speciosa
Wen‑Hsi Kuo1 · Yu‑Ling Hung2,3 · Ho‑Wei Wu2,4 · Zhao‑Jun Pan5 · Chwan‑Yang Hong6 · Chun‑Neng Wang5
© Springer Nature B.V. 2018
Correction to: Plant Cell, Tissue and Organ Culture (PCTOC) https ://doi.org/10.1007/s1124 0-018-1424-7
All hormone and antibiotic concentrations listed in the origi-nal publication were incorrect as mg/mL. They should be corrected as “mg/L”.
In the first paragraph of the Results section “Histologi-cal observation of plant regeneration”, the best shooting condition was not clear as (0.1 and 1.0 mg/mL BA). The
best shooting condition was in fact as “(0.1 mg/L NAA and 1.0 mg/L BA)”.
In the Materials and methods section “Agrobacterium-mediated genetic transformation”, the pH values of the inoculation medium and co-culture medium were not men-tioned. They should be adjusted to pH 5.2 before autoclave sterilization.
The authors apologize for these errors.
The original article can be found online at https ://doi.org/10.1007/s1124 0-018-1424-7.
* Chun-Neng Wang [email protected]
Wen-Hsi Kuo [email protected]
Yu-Ling Hung [email protected]
Ho-Wei Wu [email protected]
Zhao-Jun Pan [email protected]
Chwan-Yang Hong [email protected]
1 Institute of Ecology and Evolutionary Biology, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei 10617, Taiwan, Republic of China
2 Department of Life Science, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei 10617, Taiwan, Republic of China
3 Present Address: Institute of Plant Biology, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei 10617, Taiwan, Republic of China
4 Present Address: Institute of Plant and Microbial Biology, Academia Sinica, No. 128, Sec. 2, Academia Rd., Taipei, Taiwan, Republic of China
5 Department of Life Science, Institute of Ecology and Evolutionary Biology, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei 10617, Taiwan, Republic of China
6 Department of Agricultural Chemistry, National Taiwan University, No.1, Sec. 4, Roosevelt Rd., Taipei 10617, Taiwan, Republic of China
Vol.:(0123456789)1 3
Plant Cell, Tissue and Organ Culture (PCTOC) https://doi.org/10.1007/s11240-018-1424-7
ORIGINAL ARTICLE
Shoot regeneration process and optimization of Agrobacterium-mediated transformation in Sinningia speciosa
Wen‑Hsi Kuo1 · Yu‑Ling Hung2,3 · Ho‑Wei Wu2,4 · Zhao‑Jun Pan5 · Chwan‑Yang Hong6 · Chun‑Neng Wang5
Received: 2 January 2018 / Accepted: 13 May 2018 © Springer Science+Business Media B.V., part of Springer Nature 2018
AbstractThe florist’s Gloxinia, Sinningia speciosa, which bears considerable flower trait variations, is an emerging model plants to study floral traits development. However, the investigation of the genetic information linking these floral traits is limited due to a lack of a reliable and efficient transformation system for functional studies. This study aims to optimize a stable genetic transformation system for S. speciosa. Detailed regeneration process and tissue culture parameters are also elucidated. The results show that the plant regeneration, initiated from a single perivascular parenchyma cell, can be induced from leaf and petiole explants in the presence of 1 mg/mL 6-benzylaminopurine (BA) and 0.1 mg/mL naphthalene-acetic acid (NAA) through embryogenesis. In the presence of 0.1 mg/mL NAA only, the adventitious roots form prior to the re-differentiation of shoot tissues in leaf explants. When the proximal end of the petiole is orientated upright with the distal end to the medium, it results in higher success of regeneration, suggesting that hormone supplies must follow endogenous basipetal auxin polarity. Using a glucuronidase (GUS) reporter gene construct, maximum transformation (3.13%) was obtained after a 3 day pre-culture and 5 day co-culture from cotyledons and leaves of 3-week-old seedlings inoculating Agrobacterium strain EHA105. The putative transgenic lines were validated by RT-PCR, Southern blotting and GUS activity. Our result demonstrates that young seedlings are the best material for transformation, probably because young leaves are only a few cell layers thick allowing inner perivascular cell (the origin of regeneration) to be more accessible for Agrobacterium infiltration.
Keywords Sinningia speciosa · Tissue culture · Embryogenesis · Genetic transformation · Petiole regeneration · Competence
AbbreviationsBA 6-BenzylaminopurineNAA Naphthalene-acetic acid
Introduction
Sinningia speciosa (Lodd.) Hiern, a popular ornamental plant, is well-known for its large sized flowers, a wide range of floral color for cultivations, and frequent varieties in flo-ral shape and corolla patterning (Zaitlin and Pierce 2010). The native S. speciose has nodding, bilaterally symmetrical, bell-shape corollas with white, pink or purple colors favored by bee pollinators (Citerne and Cronk 1999; Perret 2001; Zaitlin 2012). After the first collection from Brazil in 1815, considerable cultivars of floral traits, for example, giant acti-nomorphic flowers with erect floral tubes, multiple whorls of petals, and elaborated color patterns, have been artificially selected among horticultural industry over the past century (Clayberg 1975; Citerne and Cronk 1999; Zaitlin 2012; Wang et al. 2015). In addition to their high economic values, their rapid response to artificial selection on floral traits and availability of draft genome (Aureliano Bombarely, personal communication 2016) also makes S. speciosa an emerging model plant to study the genetic changes underlying different
Communicated by Sergio J. Ochatt.
Wen-His Kuo and Yu-Ling Hung have contributed equally to this manuscript.
Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s1124 0-018-1424-7) contains supplementary material, which is available to authorized users.
* Chun-Neng Wang [email protected]
Extended author information available on the last page of the article
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floral traits (Zaitlin and Pierce 2010; Zaitlin 2012; Wang et al. 2015).
Species in genus Sinningia are especially well known for their various flower shapes used to attract a diversity of pol-linators including birds, bees, moths and bats (Perret 2001). This intragenic floral diversity is likely to constitute a great potential for developing different floral traits in horticul-tural markets. Understanding how these genetic changes are responsible for floral trait variation could provide insights into the evolution of floral traits and pollination syndromes in natural populations (Citerne et al. 2000; Perret 2001; Perret et al. 2007). It also helps to uncover how traits were genetically selected during horticultural domestication.
Agrobacterium-mediated genetic transformation is a very useful technique because the capability of manipulating a target gene aids scientists in establishing the causal relation-ship between genes and their phenotypes. Although there are few successful Agrobacterium-mediated genetic transforma-tion works published for S. speciose (Li et al. 2013; Zhang et al. 2008), no detailed and reproducible procedures were provided in their protocols and no transformation rates were mentioned. To build a reliable and efficient Agrobacterium-mediated genetic transformation system, detailed regenera-tion process is essential because the tissue competence for regeneration needs to be optimized and the accessibility of different tissues for Agrobacterium infection needs to be evaluated. In addition, an efficient transformation proto-col can be more successfully established if the ontogeny of regenerating shoot development is well-studied. In recent years, in vitro culture of S. speciosa has been extensively studied. It is mostly on the combination of auxin and cyto-kinin on the effect of shoot regeneration using leaves, stem segments or shoot apex as explants (Wuttisit and Kanchan-apoom 1996; Scaramuzzi et al. 1999; Nhut et al. 2006; Pang et al. 2006; Xu et al. 2009; Chae et al. 2012). However, these studies do not provide ontogenetic details on which tissue the plant regeneration initiates, whether the process origi-nates from a single or multiple cells, or whether the plant regenerates through embryogenesis or organogenesis.
Thus, the purpose of this study is to histologically exam-ine the regeneration processes of S. speciosa, not only by finding an optimized hormone condition for the tissue cul-ture protocol, but also by characterizing the morphogenesis of regenerated shoots. In addition, we also examined sev-eral parameters involving in the efficiency of transformation: explant sources, Agrobacterium strains, pre-culture time, and co-culture time. Finally, we characterized and report the successful stable transformation lines and their associated transformation rates.
Materials and methods
Aseptic seedling preparation
The seeds of S. speciosa (Gloxinia Avanti F1 hybrid, Blue with White Edge, SAKATA®) were sterilized by 70% etha-nol for 30 s followed by 1% sodium hypochlorite solution for 10 min. Seeds were then washed with sterilized water for at least five times and sowed on 1/2 MS medium (Murashige and Skoog 1962) with 3% sucrose, solidified with 0.3% Phytagel™ (Sigma) in a petri dish. 30 days later, seedlings were transplanted to 9 cm diameter, 11 cm height glass jars containing the same medium and sealed with the surgery tape. After 1–2 months, the aseptic seedlings were ready as the source of the explants. All the aseptic plant materi-als were kept in an incubator at 27 °C with 50% relative humidity, and placed under cool white fluorescent light at 40–60 µmol m−2 s−1 with a photoperiod of 16 h light and 8 h dark.
Tissue culture
To optimize the conditions for shoot regeneration of S. speci-osa, different plant hormones combinations were examined. 1 × MS medium (Murashige and Skoog 1962) supplemented with naphthalene acetic acid (Sigma) and 6-benzyladenine-pruine (Sigma) and 0.3% Phytagel™ (Sigma) were auto-claved (121 °C, 20 min) prior to pouring into the petri dish (50 mL medium in a 9 cm diameter, 2 cm height plate). To eliminate the size effect of the explants, uniform leaf explants were obtained by using a hole puncher (6 mm diameter). Petioles were cut into 5 or 2.5 mm pieces. There were 12 leaf explants or 9 petiole explants in each petri dish with four replicates in each treatment. The observations were recorded from 30 to 65 days after culture initiation. In each record, two data were carefully recorded: propor-tion of regenerated explants (number of responsive explants/number of total explants) and shoots per responsive explant (total number of shoots/number of responsive explants). All cultures were incubated under the same physical conditions as described above.
Paraffin sections
The samples were fixed for 6 h in FAA solution (50% etha-nol: formalin: acetic acid = 90:5:5; v/v/v) or 0.1 M phos-phate buffer (pH 7.0) with 4% paraformaldehyde and 2.5% glutaldehyde. During the fixation, the samples were kept in a vacuum oven (about 40 cmHg) to help infiltration. Then the samples were dehydrated via t-butanol series and finally into pure t-butanol and were then embedded into paraffin block.
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Continuous sections with 10 µm thick were cut by a rotary microtome (Microm, Germany). The sections were stained with safranin-O overnight and then counter-stained with fast green. After mounting with DPX (Sigma), sections were observed under a light microscope (BX51, Olympus, Japan).
Scanning electron microscope (SEM)
SEM analysis was performed with a FEI Inspect S50 micro-scope. Samples were fixed for 6 h in 0.1 M phosphate buffer (pH 7.0) with 4% paraformaldehyde and 2.5% glutaralde-hyde (at about 40 cmHg low pressure chamber). The sam-ples were then dehydrated via ethanol series and finally into pure ethanol. Ethanol was then replaced by acetone for fur-ther CO2 substitution. After coating with gold particles, the samples were ready for SEM observation.
Agrobacterium‑mediated genetic transformation
The Agrobacterium stain GV3101 and EHA105 harbor-ing the vector pCambia2301 were cultured in 3 mL YEP medium with 50 mg/mL kanamycin (for GV3101, additional 10 mg/mL gentamycin was added) overnight at 28 °C and shaking at 200 rpm. Next day, 100 µL of culture was added into 50 mL AB-MES medium (17.2 mM K2HPO4, 8.3 mM NaH2PO4, 18.7 mM NH4Cl, 2 mM KCl, 1.25 mM MgSO4, 100 µM CaCl2, 10 µM FeSO4, 50 mM MES, pH 5.5) with 20 µM acetosyringone, 0.5% glucose (m/v) and 50 mg/mL kanamycin (for GV3101, additional 10 mg/mL gentamycin was added). The culture was incubated for 20–24 h at 28 °C with shaking at 200 rpm until the OD600 reached 0.8–1.0. The cultures were then transferred into 50 mL centrifuge tube and centrifuged at 8000×g for 5 min at 4 °C. After discarding the supernatant, the Agrobacterium cells were resuspended in 50 mL inoculation medium (1 × MS medium with 3% sucrose, 200 µM acetosyringone and 0.5% glucose).
The leaf explants (5 mm2), petiole explants (5 mm long segment) and 2, 3 and 4 week-old seedlings (in toto) were prepared from aseptic culture. If pre-culture was applied, the explants were firstly placed on the pre-culture medium (1 × MS medium with 3% sucrose, 1 mg/mL BA and 0.3% Phytagel™) with a photoperiod of 16 h light and 8 h dark. For inoculation, leaf and petiole explants were soaked in the Agrobacterium for 15–20 min. For seedlings, the seed-lings and Agrobacterium were transferred into a 20 mL glass bottle. The bottle was then placed into a 50 mL syringe barrel. Additional pressure was added by push-ing the plunger until the rubber piston contacted the rim of the sample bottle. The procedure was repeated two to four times. After the inoculation, the explants were blotted dry on the sterile filter paper and transferred to co-culture medium (1 × MS medium with 3% sucrose, 0.1 mg/mL NAA, 1 mg/mL BA, 200 µM acetosyringone, 0.5% glucose
and 0.3% Phytagel™) with a photoperiod of 16 h light and 8 h dark at 27 °C. For leaf explants, the adaxial side was in contact with the co-culture medium. For petiole explants, the explants were placed horizontally on the co-culture medium.
To evaluate the transient expression efficiency, the GUS activity was measure by a chromogenic method (Jefferson 1987) after the co-culture. To localize the exact position of signals, the explants with positive signals were fixed and de-coloration in FAA (50% ethanol:formalin:acetic acid = 18:1:1) solution for 6 h. After the fixation, the sam-ples were photographed directly under the light micro-scope or continued with histological analysis by paraffin sections.
For establishing stable transgenic lines, after the co-culture, the explants were clean up by sterilized water and blotted dry on filter paper. Then, the seedlings were transferred into a 250 mL flask with 50 mL washing buffer (1/2 × MS medium with 3% sucrose and 300 mg/mL cefotaxime) and cultured at 27 °C in dark with shak-ing 125–150 rpm for 2 h to fully remove Agrobacterium. This step was repeated for three times with fresh washing buffer added additional 100 mg/mL cefotaxime each time. The cotyledon and the first pair of primary leaves of seed-lings were cut apart and attached to the selection medium (1 × MS medium with 3% sucrose, 0.1 mg/mL NAA, 1 mg/mL BA, 0.3% Phytagel™, 200 mg/mL cefotaxime and 100 mg/mL kanamycin). Then, the explants were sub-cultured every 1–2 weeks. After 12–28 weeks of selection, the regenerated shoots were cut at the base of the shoot to avoid including the original explant. The shoots were transferred to antibiotic-free medium for root induction (1 × MS medium with 3% sucrose, 0.1 mg/mL NAA, 0.3% Phytagel™, 200 mg/mL cefotaxime). After 4–6weeks, the rooted plants were transferred into pot and covered with a plastic bag for acclimation. The transformed shoots were the validated by RT-PCR for NPTII and GUS genes. No more than one transformed shoot was counted from an explant to ensure independence.
Nucleic acid isolation
Genomic DNA was isolated from 1 cm2 young leaves by cetyltrimethylammonium bromide (CTAB) method (Rogers and Bendich 1985). Total RNA was isolated by TRIzol® Reagent (Invitrogen). Additional phase separation by acid phenol: chloroform (5:1, pH 4.5) was carried out before the precipitation step in order to eliminate remaining DNA and protein contaminations. Complementary DNA was synthe-sized by M-MLV reverse transcriptase (Invitrogen) with oligo (dT)18 primer. All the procedures followed the manu-facturers’ instructions.
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RT‑PCR
Reverse transcription polymerase chain reaction (RT-PCR) was used to determine the expression of GUS gene and NPTII gene expressions in the putative transgenic lines. GUS gene was amplified by the primers GUS_800_F 5′-AGA CTG TAA CCA CGC GTC TG-3′ and GUS_1560_R 5′-ATG GTA TCG GTG TGA GCG TC-3′. NPTII gene was amplified by the primers NPTFo 5′-TCA GAA GAA CTC GTC AAG AA-3′ and NPTBaR 5′-AAC AAG ATG GAT TGC ACG CA-3′. Actin gene was used as a control gene by primers SsACT F 5′-TCC AGC AGC TTC CAT TCC TAT C-3′ and SsACT R 5′-CCA TAA ATT GCG TGT TGC TCC TGA G-3′. The PCR amplification was carried out using the following condition: 94 °C, 5 min, 94 °C, 30 s, 55–60 °C, 30 s, 72 °C, 1 min (40 cycles), 72 °C, 10 min.
Southern blotting
Southern blotting was used to confirm T-DNA insertions of the stable transgenic lines. The probe was designed as a 551 bp fragment inside the GUS gene (Supplementary Fig. 1) and synthesized by PCR DIG Probe Synthesis Kit (Roche) with primers GUS_F_1379 5′-CTT ACA GGC GAT TAA AGA GCT GA-3′ and GUS_R_1929 5′-TGA AGA TCC CTT TCT TGT TAC CG-3′. 20 µg genomic DNA was digested with EcoRI and HindIII and fractionated on a 0.8% agarose gel. The following procedures followed Roche’s DIG Application Manual for Filter Hybridization.
Results
Tissue culture conditions and plant regeneration
To obtain the best regeneration condition in S. speciosa, leaf and petiole explants were placed on the medium with hormones combinations in different concentrations. The optimized hormone combination for shoot regeneration was 0.1 mg/mL NAA together with 1.0 mg/mL BA in both
leaf and petiole explants, the proportion of regenerated explants is up to almost 90 and 60% in leaf and petiole, respectively (Table 1). In the presence of only 0.1 mg/mL NAA, the shoot regenerated through an intermediate root-ing stage (Fig. 1a, b). While in the presence of both NAA and BA (0.1 mg/mL NAA, 1 mg/mL BA; 0.1 mg/mL NAA, 2 mg/mL BA; 0.1 mg/mL NAA, 3 mg/mL BA), the shoots regenerated directly from the explants (Fig. 1c–h). For leaf explants in all hormone conditions, after 7 days of culture, the leaf explants expanded their sizes about two to four times with edges curling toward abaxial side and swelling, especially at the cut of secondary veins while cutting edges of petiole explants only swelled slightly. After 14 days of culture, in the treatment with 0.1 mg/mL NAA only, several adventitious roots formed at the edge of both leaf and petiole explants. For petiole explants, as in Fig. 1b, the adventitious roots formed exclusively at the proximal end of the peti-ole (the site attaching to the stem). While in the treatment with both NAA and BA (0.1 mg/mL NAA, 1 mg/mL BA; 0.1 mg/mL NAA, 2 mg/mL BA; 0.1 mg/mL NAA, 3 mg/mL BA), no root formation could be seen. After 30 days of culture, visible shoots appeared on the explants among all treatments, and proportion of regenerated explants and num-ber of shoots per responsive explant were carefully recorded to 65 days, by which the shoot regeneration had reached a stationary stage (Fig. 2).
For leaf explants, this treatment had almost 90% propor-tion of regenerated explants (Fig. 2a) with 2.5–3 shoots per responsive explant at the end of culture (Fig. 2a); for petiole explants, up to 50% proportion of regenerated explants with 1.5–2 shoots (Fig. 2b) per responsive explant was obtained (Fig. 2b). The treatment with 0.1 mg/mL NAA, 0 mg/mL BA (black circle) and the treatment with 0.1 mg/mL NAA, 2 mg/mL BA (black triangle) had similar performances, 10–30% lower than the proportion of the best condition (Fig. 2; Table 1). The treatment with 0.1 mg/mL NAA, 3 mg/mL BA (white triangle) had the lowest regenerative response.
While 0.1 mg/mL NAA was not the best shoot regenera-tion response, this treatment gave rise to the largest well-developed shoots and established roots, which were lacking
Table 1 Regeneration rate and efficiency of leaf and petiole explants after 65 days of culture
The regeneration rate (number of responsive explants/number of total explants) and efficiency (total num-ber of shoots/number of responsive explants) are indicated as mean ± SE, respectively. There were 12 leaf explants or 9 petiole explants in each plate with 4 replicates in each treatmenta Root-first regeneration
Hormone condition Leaf explant (n = 4 plates) Petiole explant (n = 4 plates)
NAA (mg/L) BA (mg/L) Rate (%) Efficiency Rate (%) Efficiency
0.1 0 66.7 ± 12.3a 2.17 ± 0.40 22.2 ± 17.0a 1.00 ± 0.000.1 1 86.1 ± 2.8 2.78 ± 0.21 55.6 ± 19.2 1.74 ± 0.120.1 2 79.2 ± 12.5 2.04 ± 0.42 22.2 ± 6.4 1.56 ± 0.290.1 3 31.3 ± 7.1 1.48 ± 0.19 2.8 ± 2.8 1.00 ± 0.00
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in the other treatments (Fig. 1a). The regenerative shoots from this hormone condition could be directly transferred to pot without the rooting step. However, this hormone con-dition was ineffective for petiole explants, which halted at early rooting stage and gave only few dwarf and bleached adventitious shoots at the end of the culture (Fig. 1b).
For root induction, the regenerative shoots were cut from the base to avoid contamination of the original explant. Then the shoots were placed into the hormone-free medium for rooting about 3–4 weeks. The medium was carefully washed, and the rooted plantlets were trans-ferred into pots, covering with plastic bags to reduce evaporation. After 1–2 weeks acclimation, the plastic bags were removed and cared as usual plants.
Fig. 1 Pictures of shoot regen-eration after 65 days culture. a, c, e, g Shoot regeneration from leaf explants; b, d, f, h shoot regeneration from petiole explants in horizontal orienta-tion. These petioles are placed in media with proximal end at right and distal end at left. Scale bar, 3 cm
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Effects of petiole explant orientation on shoot regeneration
In Fig. 1b, we observed the root regenerated only at the proximal end of a petiole explant. To determine whether the endogenous hormone gradient affects shoot regeneration, different orientations in which the petiole explants attach to the culture medium were tested. Figure 3 shows that the inverted placement of petioles with distal end (the site to leaf blade) inserted in the medium allowed the explants to regenerate more shoots (black circle in Fig. 3a, b; also
see photo in Fig. 3c) than that with proximal end attached (white circle in Fig. 3a, b; also see photo in Fig. 3d). This dramatic difference could not be seen when we horizontally placed petiole explants on the medium, the newly regener-ated shoots emerged out from both the proximal and the distal ends (Fig. 3e). To evaluate the petiole explants size on successful regeneration, we further cut the petiole explants into half-length (2.5 mm) and inversely placed petioles into the medium. The proportion of regenerated explants reduced into half compared to that of full-length petiole (white tri-angle in Fig. 3a), but the number of shoots per responsive
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Fig. 2 The effects of BA and NAA on shoot regeneration from leaf and petiole explants. a Shoot regeneration from leaf explants; b shoot regeneration from petiole explants. Proportion of regenerated explants is defined as number of responsive explants (the shooting explants) divided by total explants. Shoots per responsive explant
is defined as total number of regenerative shoots divided by total responsive explants. The data are presented as mean ± standard error with four replicates. Each replicate has 12 leaf explants or 9 petiole explants
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explant remained approximately the same as the original one (white triangle in Fig. 3b). To summarize these experiments, we conclude that the orientation (especially when placed vertically on medium) and size of petiole is very important for shoot regeneration.
Histological observation of plant regeneration
A detailed histological examination was done for the best shooting condition (0.1 and 1.0 mg/mL BA). Before the ini-tiation of regeneration, the leaves composed of 2 layers of
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licates and each replicate has nine petiole explants (2.5 mm petiole treatment has 12 petiole explants); c–f pictures of shoot regeneration after 65 days culture from each of above petiole orientations
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Fig. 4 Shoot regeneration progress of leaf explants. a Leaf section 2 layers of palisade parenchyma (pa) are indicated; b initiation of shoot showing meristematic activity from one perivascular parenchyma cell, characterized by large nuclei and dense cytoplasm with continu-ous cell divisions without cell elongation; c somatic embryo at early globular stage. suspensor (su); d callus (ca) formation near the cut-ting site with several meristematic tissues (me) scatting around the
vascular bundles; e–h somatic embryo at late globular stage (e), heart stages (f, g) and cotyledonary stage (h). ap Apical meristem, ro root pole, st starch grains, pr procambium tissue; i starch grains found in somatic embryo cells at cotyledonary stage; j well-developed api-cal meristem of a regenerated plant; k, l morphology of regenerated shoots on leaf explants (by dissecting microscope and SEM). ea early stage, la late stage
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palisade mesophyll tissue (“pa” in Fig. 4a) (the lower one was not fully differentiated), 4–5 layers of sponge mesophyll tissue and a single layer of epidermis tissue at each side of the leaf. After 14 days of culture, several dome-shaped structures appeared at the edge of the leaf explant (cross sec-tion in Fig. 4d, and outside morphology in Fig. 4k, marked as “ea”). When sectioning these dome-shaped structures, as shown in Fig. 4d, the upper epidermis and sponge mesophyll tissue underwent continuous cell divisions and enlargement, giving rise to several layers of callus cells (“ca” in Fig. 4d). Several meristematic tissues (“me” in Fig. 4d) with dense cytoplasm and prominent stained nuclei, can be seen around the proliferated vascular tissues. Figure 4b shows that these meristematic tissues originated from a single perivascular parenchyma cell, which underwent continuous cell divisions without cell expansion. Then, accompanied by repeated cell divisions and expansions, the meristematic cells formed a globular embryogenic callus (Fig. 4c, e). Note that a suspen-sor-like structure (denoted as “su”) can be seen in Fig. 4c. The globular embryogenic callus continued to differentiate into spindle-shape (Fig. 4f). In this stage, the polarity had been established as the presence of both shoot apical mer-istem (“ap” in Fig. 4f) and root pole (“ro” in Fig. 4f). This pre-embryonic callus continued to differentiate into triangu-lar or heart shape (Fig. 4g), and finally to a cotyledon-like stage (Fig. 4h). At cotyledonary stage, the matured somatic embryo fragmented and isolated from the explant (Fig. 4h). In particular, many large starch grains, distributed within the cells in the transition zone between the root pole and the apex regions, were observed (“st” in Fig. 4h and close look in Fig. 4i). Procambial strands, extending from coty-ledons into the hypocotyl, were also seen (“pr” in Fig. 4h). Finally, the regenerated shoot protruded out from the explant and well-established shoot apical meristem was observed (Fig. 4j, dissecting microscope view in Fig. 4k; scanning electronic microscope view in Fig. 4l).
Optimization of Agrobacterium‑mediated transformation
Based on the observation that the regeneration is derived from a single perivascular parenchyma cell, we realized that the tissue barrier of surrounding mesophyll cells could hinder the Agrobacterium-mediated transforma-tion. Therefore, we turned to S. speciosa young seedlings with simpler leaves structure to facilitate Agrobacterium-mediated transformation. To obtain a reliable transforma-tion system, GUS enzymatic activity was used to evaluate the transformation rate among different treatments. Pic-tures of successfully transformed seedlings showed that the GUS signals distributed in both cotyledons and the first pair of primary leaves, especially around the wounded sites (Fig. 5a–c). In addition, paraffin sections show more
precise locations of the transformed cells. By adjusting the focusing plane of the microscope, it was confirmed that the transformed tissues revealed by GUS signals were located at the mesophyll cells, epidermis cells and the glandular trichome head cell of the cotyledon (Fig. 5d–f). Various conditions were tested in a step-by-step manner, including explant sources, co-culture time, Agrobacte-rium strains, age of seedlings and pre-culture time. The results show that the seedlings (54.6%) had much better transient expression rate compared to leaves (13%) and petioles (2%) (Fig. 5 and Supplementary Fig. 2). There-fore, seedlings were chosen for Agrobacterium-mediated transformation in the following experiments.
For seedlings, Agrobacterium strain EHA105 had better performance than the strain GV3101 (Fig. 5g, h). Longer co-culture time, up to 5 days, could promote higher trans-formation rate (Fig. 5 g). When co-culture time extended to 6 or 7 days, the Agrobacterium can not be eliminated; similarly, if the co-culture time is < 3 days, no seedling can be infected (data not shown). Under the condition of 5-days co-culture with Agrobacterium strain EHA105, 4-week-old seedlings had greater transient expression rate than those of 2- and 3-week-old seedlings (Fig. 5 h). However, the GUS signals in 3-week-old seedlings were the strongest in the all treatments (Supplementary Fig. 3). Therefore, 3-week-old seedlings were chosen as the explant sources in the following stable transformation treatments. Finally, longer pre-culture time (4 days) could also enhance the transient expression rate (Fig. 5i).
Characterization of stable transformation lines
To obtain stable transformation lines of S. speciosa, 192 three-day pre-cultured seedlings were inoculated with Agrobacterium. Of these, 50 seedlings gave rise to shoots after 12–28 weeks of selection. Six were confirmed as successful transformants through RT-PCR (6/192 = trans-formation rate 3.13%, Fig. 6a). After root induction and transplanting into pots, putative transgenic plants were tested for their GUS activity in their young leaves and sepals or anthers (Fig. 6b). The plants with positive GUS activity were further confirmed by RT-PCR (Fig. 6c) and Southern blotting (Fig. 6d). The plants with both GUS gene and NPTII gene expression in RT-PCR were docu-mented as successful transgenic lines. Southern blotting was done for three out of six independent lines (Fig. 6c). One transgenic lines (Line 2) showed a single insertion event while the other two transgenic lines (Line 1 and 4) showed two insertion events. The results above show that our protocol could successfully introduce foreign genes into the genome of S. speciosa.
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54.6%
76.4%83.3%
0.0% 1.7% 0.0%
2 weeksn = 11
3 weeksn = 50
4 weeksn = 10
EHA105
GV3101
Age of Seedlings
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ion
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20
40
60
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ion
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20
40
60
80
100
3 daysn = 20
5 daysn = 11
Co-culture time
EHA105
GV3101
0 day 3 days 4 days
Tran
sien
t exp
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ion
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0
20
40
60
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78.33% 85.71%
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Discussion
In this paper, we found that step-by-step optimization and shoot ontogeny are important factors for acquiring a sat-isfying transformation system for S. speciosa, particularly because details on these factors and transformation rates were not reported from previous protocols (Zhang et al. 2008; Li et al. 2013). From our results, we achieved an effi-cient and stable genetic transformation system for S. spe-ciosa by carefully examining the best hormone combina-tions (0.1 mg/mL NAA and 1 mg/mL BA) and which tissue (perivascular parenchyma cells) had the best attained com-petence for shoot regeneration. Under these optimal in vitro culture conditions, we have shown Agrobacterium strain EHA105 infection on cotyledons and leaves of 3-week-old seedlings give the highest transformation rates at 3.13%. We demonstrated that tissue with 3-day pre-culture and 5-day co-cultivation also significantly facilitated the Agrobacte-rium infection. These reliable transformation procedures can be applied to further functional studies to understand the genetic control of floral traits. For S. speciosa, in particular, the association of several floral traits and candidate genes such as CYCLOIDEA has been identified. (Hsu et al. 2015, 2017; Wang et al. 2015). With the new input of draft genome information (Aureliano Bombarely, personal communication 2016), candidate genes associated in floral phenotype can be causally examined by functional studies with our transfor-mation procedures.
Hormone conditions for regeneration
The factors to achieve successful in vitro tissue regeneration include exogenous hormone conditions, tissue orientations in response to internal hormone gradient, as well as regen-eration cell type, developmental stages, and the age of the explants (Gilissen et al. 1996). For example, the regeneration success in tobacco explants depends on cellular location, cell cycle phase and ploidy level (Gilissen et al. 1994).
Our results indicated that the hormone conditions at 0.1 mg/mL NAA with 1 mg/mL BA yielded the optimal
regeneration rate (86.1 ± 2.8%) from leaf explants. This rate is similar to the previous report on another cultivar of S. speciosa that proportion of regenerated explants can be as high as > 90%, although the best hormone combination they found was 0.2 mg/mL NAA with 2 mg/mL BA (Xu et al. 2009). Nonetheless, both our findings and previous reports concluded that the effect of combination of NAA and BA is much suitable for S. speciosa regeneration than the combina-tion of indole-3-acetic acid and kinetin or the combination of indole-3-acetic acid and BA (Scaramuzzi et al. 1999). In addition, these seem to indicate that the ratio of NAA to BA equal to 1:10 is crucial for the effective shoot regeneration in S. speciosa rather than their actual concentration.
Our study also found that certain hormone combinations can trigger shoot-first or root-first regeneration. Combining hormone NAA and BA could induce shoot-first regeneration, but NAA could only induces root-first regeneration from leaf explants. It is worth to mention that the leaves of root-first regeneration (Fig. 1a) are larger and morphologically health-ier than that of the shoot-first ones (Fig. 1c). Furthermore, root-first explants save 3–4 weeks of regeneration time pos-sibly because these roots help to absorb nutrients efficiently from medium. Based on both our findings and Xu et al. (2009), root-first regeneration could be more suitable for in vitro culture of S. speciosa. It should be noted that higher concentrations of NAA are not favorable because it represses shoot regeneration after the rooting stage (Murashige 1977).
Petiole as explant source for regeneration
Petiole has been considered an alternative source of the explants for micro-propagation. Our result is the first report for investigating its competence to cytokinin (BA) and auxin (NAA) in S. speciosa. In other Gesneriaceae species, such as African violet and Primulina dryas, the petiole’s response to hormones were investigated in which petiole is less com-petent for regeneration than leaf explants do (Mithila et al. 2003; Padmanabhan et al. 2015). Although our data also suggested the proportion of regenerated explants and the number of shoots were lower from petioles than from leaves (Figs. 1, 2), the cut ends of petioles, however, have more perivascular tissues exposed which could facilitate Agro-bacterium infection. In particularly we found that regen-erated shoots were derived from perivascular parenchyma cells. Our results reveal that the petiole regeneration rates in response to hormones such as 0.1 ppm NAA only and 0.1 ppm NAA + 1 ppm BA have large standard error than those in higher amount of BA (Fig. 2b). This could be explained by that low BA supply, although it is likely to induce more petiole regenerations, the chance is not stable. Given the fact that the regeneration rates from petiole are lower than that from leaves and the rates vary a lot, leave could be a better regeneration material.
Fig. 5 Transient GUS expression in seedlings of S. speciosa. a–c Pic-tures of transformed seedlings. GUS signals present in the success-fully transformed cells. Note that there are several transformed cells around the wound, which was made by forceps during inoculation (arrow in b); d–f paraffin sections of transformed seedlings. Note that the GUS signals distribute in epidermis, mesophyll, vascular tissues and a glandular trichome head cell; g–i effect of co-culture time (g), age of seedlings (h) and pre-culture time (i) on transient expression rate in S. speciosa from the comparisons of Agrobacterium strain EHA105 and GV3101. The data (i) are presented as mean ± stand-ard error with four repeated experiments at least, 30–50 seedlings were used in each experiment. Transient expression rate = (num-ber of explants with positive signals)/(total number of treated explants) × 100%
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21226
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)
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b
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EcoRI HindIII EcoRI HindIII EcoRI HindIII EcoRI HindIII EcoRI
1 2 3 4 5 6 WT
GUS (783 bp)
NPTII (788 bp)
Actin (550 bp)
1 2 3 4 5 6 WT plasmid Neg
1 2 4 WT plasmid
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Similar to African violet (Mithila et al. 2003), the pres-ence of exogenous cytokinin is necessary for S. speciosa to induce somatic embryos in petiole explants; otherwise they remain suspended at rooting stage (Fig. 1b). In contrast, the leaf explants could regenerate in the presence of auxin (NAA) only (Fig. 1a). Interestingly, both leaf explants and petiole explants did not regenerate when the presence of only artificial cytokinin (BA) (supplementary Fig. 4), indicating that exogenous auxin (NAA) is also essential for the plant regeneration from both leaf and petiole explants.
Effect of petiole orientation on regeneration
Effects of explant orientation have been reported an impor-tant factor on successful shoot regenerations in flowering plants as well as in Gesneriaceae species (García-Luis et al. 1999; Bhatia et al. 2005; Ma et al. 2010; Padmanabhan et al. 2015). Most of these studies examined whether specific tis-sue orientation such as leaf with abaxial or adaxial site in contact with the medium can significantly affect regenera-tion success (Bhatia et al. 2005; Ma et al. 2010; Padmanab-han et al. 2015). Our result here, however, is the first report to characterize the vertical placement of petiole segments on the medium needs to be inverted (distal end down) for successful regeneration. In our vertically placed petioles, when either distal end or proximal end of the petiole ver-tically attached to the medium, only the distal end down placements can regenerate (shoots generated at the proximal end, Fig. 3c). The best explanation is that in vitro culture of petiole probably requires following endogenous auxin transport direction. It is known that leaf auxin flow moves basipetally from distal to proximal end inside the petiole. Therefore, only when distal end of the petiole attached to the medium (BA + NAA), the exogenous hormone supply direction coincides with endogenous auxin flow direction allowing successful shoot regeneration. The basipetal auxin transport has been reported as an important factor to stim-ulate the root growth whereby auxin was transported and accumulated (Estelle 1998; Leyser 2005), but seldom the auxin flow effects on petiole shoot regeneration is investi-gated. In addition, the proportion of regenerated explants
declined when the size of petiole explants was cut into half (Fig. 3a, but also true in horizontally placed petioles, data not shown), suggesting that not only the correct orientation, but also the adequate size (5 mm) of petiole explants should be taken into consideration in practical regeneration applica-tions. Because horizontally placed petioles achieved higher proportions of regenerated explants, we suggest this orienta-tion should be used for petiole regeneration.
Different tissues respond differently to hormone combinations to regenerate shoots
The shoot-first regeneration in S. speciosa was initiated from a single perivascular parenchyma cell near to leaf veins without a callus intermediate stage under 0.1 mg/mL NAA and 1 mg/mL BA (Fig. 4). However, in Scaramuzzi et al. (1999), when leaf disc of S. speciosa cultured in much higher hormone combination with 5 mg/mL IAA and 5 mg/mL BA, shoots are regenerated from callus. They found initiation of shoots starts from multiple mesophyll cells of spongy parenchyma, which is different from our results. This indicates different tissue layer of S. speciosa can response to different hormone combinations to attain competence for shoot induction.
Regeneration via somatic embryogenesis
Somatic embryogenesis is preferred as a plant regeneration system for genetic transformation because somatic embryo is usually single cell origin (Gaj 2001; Cui et al. 2009). Direct somatic embryogenesis from single cell can avoid chimeras and less likely to have somaclonal variation (Gaj 2001). In our hormone combination treatments, the regenerative tissue in S. speciosa appears to undergo somatic embryogenesis process from the perivascular parenchyma cell as a single cell origin (Fig. 4b). Particularly, a suspensor-like structure observed in shoot initiation stage of regenerating tissues (Fig. 4c) is also a convincing evidence of regeneration by embryogenesis (Ho and Vasil 1983; Nagmani et al. 1987). During the globular stage (Fig. 4c–e), although the somatic embryo is very close to the explant’s vascular bundles, there is no vascular bundle connection between old explants and regenerative tissues. In cotyledonary stage (Fig. 4h, i), starch grains were extensively detected in the transition zone of the somatic embryo, which is also a characteristic of a develop-ing embryo (Focks and Benning 1998; Baud et al. 2002). The regeneration of shoots from inside perivascular tissue may increase the difficulty of transformation because the physical barrier of surrounding mesophyll tissue blocks the accessibility of Agrobacterium. However, this problem can be circumvented by using young seedlings which are only few cell layers thick allowing successful Agrobacterium infections as our results indicated.
Fig. 6 Transformation efficiency and characterization of success-ful transgenic lines. a Regeneration and transformation rate from different pre-culture times. The data are presented as mean ± stand-ard error with four repeats, 30–50 seedlings in each experiment; b GUS activity assays in transgenic plants. GUS signals can be found in transgenic leaves, sepals and stamens (no. 1–6) with wild-type (WT) leaves as control; c RT-PCR analysis of GUS and NPTII mRNA expression in transgenic plants. ACTIN serves as the internal control; d Southern blot analysis of selected transgenic plants. 20 µg of genomic DNA was digested with EcoRI and HindIII and hybrid-ized with DIG-labeled probe containing GUS gene (551 bp). Plasmid (pCambia 2301) is used as positive control in RT-PCR and Southern blotting
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Regenerations through somatic embryogenesis are also reported in some Gesneriaceae species. In Aeschynan-thus radicans, somatic embryos regenerated directly from cut edges of leaf explants (Cui et al. 2009). In Primulina tabacum, the regenerated shoots initiated from the explants inside mesophyll layers through somatic embryogenesis when high concentration (5 µM) of Thidiazuron is used (Yang et al. 2012). Similarly, relatively high concentration (2.5 µM) of TDZ-induced somatic embryos in Saintpaulia ionantha at epidermal and sub-epidermal cell (Mithila et al. 2003). Substitute TDZ with BA (5 µM) and low concen-tration of TDZ (< 2.5 µM), instead, induced shoot organo-genesis in P. tabacum and S. ionantha, respectively. These are in line with other reports that TDZ alone or combined with other PGRs often induced somatic embryogenesis in many plant species (Murthy et al. 1998). To our surprise, we found somatic embryos can also be induced through low concentration of BA (1 mg/mL BA and 0.1 mg/mL NAA) in S. speciosa.
Establishment of Agrobacterium transformation system
In this study, Agrobacterium-mediated transformation sys-tem of S. speciosa reached to a transformation rate level at 3.13% (Fig. 6). We found factors to improve Agrobac-terium-mediated transformation in S. speciose including using 3-week-old seedlings, Agrobacterium strains EHA 105, 3-day pre-culture and 5-day co-culture. Although pre-viously several genetic transformation studies published for S. speciosa mainly used leaves for materials, those results did not provide transformation rates allowing us to com-pare which tissue is best for infection or whether our pro-tocol is more efficient or not (Zhang et al. 2008; Li et al. 2013; Wang et al. 2014). However, our result did suggest that transformation rate of seedlings is higher than that of leaves and petioles in S. speciosa (Supplementary Fig. 2). Seedling, comparing to leaf and petiole explants, usually are more susceptible to Agrobacterium because its immune response has not been fully established (Wu et al. 2014). Therefore, seedlings and cotyledons-based transformation systems are commonly exploited, for example, in tomato or soybean (Rai et al. 2012; Zhang et al. 2014). Indeed, coty-ledons of Gesneriaceae species have been reported to have prolonged meristematic activities (Nishii et al. 2013). Infec-tion on these meristematic cotyledon cells gives a higher chance to regenerate and obtain successful transformants. Furthermore, seedlings of S. speciosa have only 1–2 lay-ers of mesophyll cells (Fig. 5e), enabling the perivascular cell (the origin of regeneration) to be more accessible to Agrobacterium.
Our data showed Agrobacterium strain EHA105, which was also used in previous studies (Zhang et al. 2008; Li
et al. 2013), appears to be the most suitable agent for S. speciosa genetic transformation. To increase the Agrobacte-rium infection and subsequent successful transformation, we found pre-culture and extended co-culture time are required. It has been found that pre-culture can significantly increase the transformation rate in Gesneriacese species, such as Kohleria sp. (Geier and Sangwan 1996), Saintpaulia ion-antha (Kushikawa et al. 2002), and Chirita pumila (Liu et al. 2014).
In conclusion, we have demonstrated that S. speciosa seedlings, leaf tissues and petioles can be induced to initi-ate shoots through somatic embryogenesis under the com-bination of BA and NAA. Somatic embryos derived from a single perivascular cell origin provide better chances for getting non-chimeric transformants. With only few cell lay-ers, pre-culture seedlings serve as the best material for Agro-bacterium infiltration providing an adequate co-culture time. Our optimized tissue culture protocol and Agrobacterium-mediated genetic modification system shall offer additional tools for the generation of new varieties in S. speciosa for commercial floriculture needs.
Acknowledgements WH Kuo and YL Hung were supported by National Taiwan University Research Student Scholarship, and the main funding from Ministry of Science and Technology of Taiwan was granted to CN Wang (MOST 103-2313-B-002 -004 -MY3 and 106-2313-B-002 -035 -MY3).
Author contributions WHK, YLH and HWW participated in conduct-ing the experiments. WHK, YLH and CNW participated in analyzing and interpreting the data and writing the manuscript. ZJP participated in interpretation of the data. CYH and CNW conceived the idea and designed the experiments. CNW revised the manuscript and gave final approval of the version to be published. All authors read and approved the final manuscript.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing interests.
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Plant Cell, Tissue and Organ Culture (PCTOC)
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Zaitlin D, Pierce AJ (2010) Nuclear DNA content in Sinningia (Gesne-riaceae); intraspecific genome size variation and genome charac-terization in S. speciosa. Genome 53:1066–1082
Zhang MZ, Ye D, Wang LL, Pang JL, Zhang YH, Zheng K, Bian HW, Han N, Pan JW, Wang JH, Zhu MY (2008) Overexpression of the cucumber LEAFY homolog CFL and hormone treatments alter
flower development in gloxinia (Sinningia speciosa). Plant Mol Biol 67:419–427
Zhang F, Chen C, Ge H, Liu J, Luo Y, Liu K, Chen L, Xu K, Zhang Y, Tan G, Li C (2014) Efficient soybean regeneration and Agro-bacterium-mediated transformation using a whole cotyledonary node as an explant. Int J Appl Biotechnol Biochem 61:620–625
Affiliations
Wen‑Hsi Kuo1 · Yu‑Ling Hung2,3 · Ho‑Wei Wu2,4 · Zhao‑Jun Pan5 · Chwan‑Yang Hong6 · Chun‑Neng Wang5
Wen-Hsi Kuo [email protected]
Yu-Ling Hung [email protected]
Ho-Wei Wu [email protected]
Zhao-Jun Pan [email protected]
Chwan-Yang Hong [email protected]
1 Institute of Ecology and Evolutionary Biology, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei 10617, Taiwan, Republic of China
2 Department of Life Science, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei 10617, Taiwan, Republic of China
3 Present Address: Institute of Plant Biology, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei 10617, Taiwan, Republic of China
4 Present Address: Institute of Plant and Microbial Biology, Academia Sinica, No. 128, Sec. 2, Academia Rd., Taipei, Taiwan, Republic of China
5 Department of Life Science, Institute of Ecology and Evolutionary Biology, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei 10617, Taiwan, Republic of China
6 Department of Agricultural Chemistry, National Taiwan University, No.1, Sec. 4, Roosevelt Rd., Taipei 10617, Taiwan, Republic of China
